The present invention provides, among other things, assays and methods of diagnosis and treatment that are based on the surprising observation of an interaction between amyloid xcex2 or its aggregates with sodium channels. In particular, the present invention provides methods to identify compounds that modulate this interaction, and methods of diagnosis and treatment that are based on this interaction.
Certain neurodegenerative diseases such as Alzheimer""s disease and Down""s syndrome are characterized by the presence of insoluble aggregates of amyloid xcex2 (Axcex2). This 39-43 amino acid peptide is derived by abnormal proteolysis from the Amyloid Precursor Protein (APP). It is well known that the amyloid xcex2 aggregates/plaques widely found in the brain and intraneuronal neurofibrillary tangles of Alzheimer""s disease patients can modulate neurite outgrowth, synaptogenesis, synaptic plasticity or cause neuronal death (Mattson et al, Trends Neurosci., 16, 406-415 (1993)). The mechanism(s) by which these effects are accomplished remain elusive, however (Frazer et al, Trends Neurosci., 20, 67-72 (1997)).
Presently, one theory maintains that these deleterious effects may originate from amyloid xcex2""s ability to promote chronic calcium influx into neurons through modulation of neuronal calcium ion channels (Daidson et al., Brain Res., 643, 324-327 (1994)), or to form transmembrane cation-permeable channels (Kawahara et al., Biophysiol. J., 73, 67-75 (1997)) that indiscriminately allow calcium, sodium, and other cations to flood a cell""s interior and destroy its calcium homeostasis. Calcium channel blockers such as nimodipine that can cross the blood-brain barrier have been shown to slow the progression of Alzheimer""s disease in some patients (Tollefson, Biol. Psychiatry, 27, 1133-1142 (1990)). Similarly, zinc cation, which is known to bind at specific amyloid xcex2 1-40 sites (Bush et al, Science, 265, 1464-1467 (1994)), can block the calcium influx through amyloid xcex2 1-40 channels (Arispe et al., Proc. Natl. Acad. Sci., 93, 1710-1715 (1996)).
In addition, amyloid xcex2 aggregates have been shown to induce abnormal potassium ion channel activity. In cultured hippocampal neurons, amyloid xcex2 opens a calcium-sensitive, large conductance potassium channel (i.e., iberitoxin-sensitive BK) (Furukawa et al., Nature, 379, 74-78 (1996)), which could lead to chronic loss of cytoplasmic potassium and destroy the ability of the neurons to generate and propagate action potentials critical for brain signaling functions. Such aberrant potassium channel activity has in fact been noted in Alzheimer""s disease patients (Etchebemgaray et al., Proc. Natl. Acad. Sci., 90, 8209-8213 (1993)). Likewise, in the cultured cholinergic septal cell line SN56, amyloid xcex2 (e.g., amyloid xcex2 1-40) causes cell death by impacting a TEA-sensitive potassium channel (Colom et al., J. Neurochemistry, 70, 1925-1934 (1998)).
These studies suggest that amyloid xcex2 aggregates exert their effects through multiple targets. None of the targets that have been described, however, would result in the rapid and complete inhibition of neuronal electrical impulses critical for brain function that is observed with advanced stage Alzheimer""s disease. Furthermore, the ion channel studies described above were performed on cultured neurons, whose properties may have been changed significantly through dedifferentiation in culture conditions. Therefore, the data currently available likely does not represent the true response of native neurons.
Recent genetic studies have shown that mutations in the amyloid protein precursor and presenilin genes affect the processing and production of amyloid xcex2 and thus are related to age of onset and susceptibility to Alzheimer""s disease. Additional studies have shown that variants in ApoE genes also affect susceptibility and age of onset for Alzheimer""s disease. Spontaneous Alzheimer""s disease is hypothesized to be under the control of other genes and environmental factors that have yet to be identified.
Additionally, inhibition of amyloid xcex2 aggregation, and hence toxicity, is believed to be beneficial therapeutically in the treatment of Alzheimer""s disease. Several reports have appeared providing evidence that small, diffusable aggregates (Lambert et al., Proc. Natl. Acad. Sci., 95, 6448-6453 (1998); PCT International Application WO 98/33815), and protofibrils (Harper et al., Chem. Biol., 4, 119-125 (1997); Harper et al., Chem. Biol., 4, 951-959 (1997); Walsh et al., J. Biol. Chem., 272, 22364-22374 (1997)), and not the completely fibrillar peptide, might be the Axcex2 species toxic to living cells. The physical differences in the alleged toxic species identified by these different research groups xe2x80x9csupports the concept that different Axcex2 assemblies have distinct neurobiological activities, which may be manifested differently using an electrophysiological readoutxe2x80x9d (Hartley et al., J. Neuroscience, 19, 8876-8884 (1999)). Thus, the etiology of Alzheimer""s disease may be quite complex, and may warrant a variety of different avenues of diagnosis and treatment.
Along these lines, U.S. Pat. No. 5,876,948 describes screening methods to identify inhibitors of the neurotoxic effect of amyloid xcex2. In particular, the ""948 patent purportedly provides a method of screening candidate compounds, wherein the method comprises obtaining a cell such as a primary neuron, a neuronal cell, or a cell developmentally derived from neuronal tissue. The cell is contacted with a candidate compound in the presence of a neurotoxin selected from the group consisting of amyloid xcex21-38, xcex21-40, xcex21-43, and xcex229-35, and it is determined whether the compound reduces the effect of the neurotoxin (e.g., reduces cell death), or reduces accumulation of amyloid xcex2 on the cell surface. Accordingly, this patent provides no information on how to screen for compounds that target cell events that precede cell death, apart from accumulation of the peptide on the cell surface. A need thus remains for useful assays to screen for compounds that exert protective effects at a stage prior to cell death mediated by amyloid xcex2, and for compounds that exert their effect apart from interference with amyloid xcex2 deposition on the cell surface. Furthermore, the ""948 patent does not appear to consider any effect of the aggregation state of amyloid xcex2 on neurotoxicity. A related patent, U.S. Pat. No. 5,137,873, claims a method for treating a disease that is characterized by accumulation of amyloid xcex2, which comprises administration of a therapeutically effective amount of a tachykinin agonist such as substance P.
U.S. Pat. No. 5,892,018 pertains to DNA sequences encoding a novel subfamily of amiloride-sensitive sodium channel proteins from the human central nervous system. PCT International Application WO 98/54316 describes a new class of sodium channel protein that may function as a receptor for endogenous transmitters. Both the ""018 patent and the ""316 application suggest that ion channels may play some role in the pathogenesis or treatment of Alzheimer""s disease (see, ""316 application, pages 1 and 15; see, ""018 patent, column 1, lines 50-54). However, neither document presents any assay for diagnosis of Alzheimer""s disease, much less any methods for its treatment.
Thus, there remains a need for further understanding of the functioning of amyloid xcex2 or its aggregates, and means to diagnose, predict, prevent and treat diseases, disorders, and conditions that result from amyloid xcex2 or its aggregates. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The present invention relates to a surprising observation of an interaction between amyloid xcex2 or its aggregates with sodium channels. Based on this novel and unexpected interaction, the present invention provides a method for identifying compounds that modulate the interaction of amyloid xcex2 or its aggregates with a sodium channel, wherein the method preferably comprises contacting a cell which comprises a sodium channel with amyloid xcex2 or its aggregates in the presence and in the absence of a test compound, and then determining whether there is any impact on the sodium channel in the presence of the test compound as compared with in the absence, with a compound that exhibits an impact being considered a modulator of the interaction of amyloid xcex2 or its aggregates with said sodium channel. In preferred embodiments, the impact on the sodium channel is assessed by either examining resting membrane potential, action potential, or measuring the fast inward sodium current. Preferably as employed in the invention, the cell is a neuronal cell, or is a non-neuronal cell, as described herein.
The method of identifying compounds that modulate the interaction of amyloid xcex2 or its aggregates with a sodium channel also can be carried out according to the invention wherein the method comprises obtaining a first cell that produces amyloid xcex2 or its aggregates, obtaining a second cell comprising a sodium channel, contacting the second cell with the first cell in the presence and absence of a test compound, and determining the activity of the sodium channel in the presence of said test compound as compared with in the absence, wherein a test compound that impacts activity is considered a modulator of the interaction of amyloid xcex2 or its aggregates with said sodium channel. Preferably one of the first and second cells is a neuronal cell and the other is a non-neuronal cell, or both of the first and second cells are either a neuronal cell or a non-neuronal cell.
Also, preferably the method of identifying compounds that modulate the interaction of amyloid xcex2 or its aggregates with a sodium channel can be carried out according to the invention wherein the method comprises the steps of obtaining a composition comprising a purified sodium channel, contacting the sodium channel with amyloid xcex2 or its aggregates in the presence and absence of a test compound, and determining the activity of the sodium channel in the presence of the test compound as compared with in the absence, wherein a test compound that impacts activity is considered a modulator of the interaction of amyloid xcex2 or its aggregates with the sodium channel.
The invention also provides a method for diagnosing Alzheimer""s disease (or prion disease) in a mammal suspected of having Alzheimer""s disease (or prion disease), the method comprising the step of measuring the activity of a sodium channel in a cell of the mammal, wherein reduced activity of the sodium channel is correlated with the existence of an Alzheimer""s disease state (or a prion disease state).
The invention further provides a method of screening a mammal for susceptibility to Alzheimer""s disease, comprising the steps of:
(a) isolating a cell from the mammal, the cell comprising a sodium channel; and
(b) measuring activity of the sodium channel in the absence and in the presence of amyloid xcex2 protein;
wherein reduced activity of the sodium channel in the presence of amyloid xcex2 as compared to in the absence is correlated with a susceptibility to developing Alzheimer""s disease.
Additionally, the invention provides a method for preventing, treating, or reversing diseases such as Alzheimer""s disease and prion disease in a mammal, wherein the method preferably comprises contacting a sodium channel of the mammal with a compound that protects the sodium channel from a negative impact of amyloid xcex2 or its aggregates. This method optionally is carried out with use of a compound that opens the sodium channel.
Furthermore, the invention provides for the use of a compound that modulates the interaction of amyloid xcex2 or its aggregates with cellular sodium channels for the manufacture of a medicament for the treatment of a neurological disorder (e.g., Alzheimer""s disease).
Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be recombined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.
In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.